title: Insights into the conformation of aminofluorene-deoxyguanine adduct in a DNA polymerase active site.
abstract: The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo(-)) and DNA polymerase β (pol β) using (19)F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol β. The addition of a non-hydrolysable 2'-deoxycytosine-5'-[(α,β)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo(-) complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with (19)F NMR data. Surface plasmon resonance binding kinetics revealed that pol β binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential.
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, USA.
authors.forename: Vaidyanathan G
authors.initials: VG
authors.lastname: Vaidyanathan
authors.forename: Fengting
authors.initials: F
authors.lastname: Liang
authors.forename: William A
authors.initials: WA
authors.lastname: Beard
authors.forename: David D
authors.initials: DD
authors.lastname: Shock
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
authors.forename: Bongsup P
authors.initials: BP
authors.lastname: Cho
mesh_terms: D020134:Catalytic Domain; D018736:DNA Adducts / Q000737:chemistry*; D004256:DNA Polymerase I / Q000737:chemistry*; D019951:DNA Polymerase beta / Q000737:chemistry*; D004261:DNA Replication; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D005449:Fluorenes / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
chemical_list: D018736:DNA Adducts; D005449:Fluorenes; C031838:N-(deoxyguanosin-8-yl)-2-aminofluorene; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; D003849:Deoxyguanosine
keywords: DNA Damage; DNA Replication; DNA Structure; Mutagenesis Mechanisms; NMR